The invention concerns single-stranded oligodeoxynucleotides, certain derivatives thereof and methods of their use for introducing a predetermined change at a predetermined location in a target gene in a living cell. The cell can be a mammalian, insect, fish, worm or avian cell, either in an artificial culture medium or in an organism, a bacterial cell or a plant cell. The target gene can be a chromosomal gene or an extrachromosomal gene, i.e., on a bacterial artificial chromosome.
Techniques of making a predetermined change at a predetermined location in a target nucleic acid sequence of a cell have been described. These techniques utilize the cell""s enzymes that concern DNA repair and homologous recombination. In these techniques an oligonucleotide or oligonucleotide analog is synthesized that contains two regions that have the sequence of the target gene that flank a region, termed a xe2x80x9cmutator regionxe2x80x9d, that differs from the target gene. In this application such oligonucleotides and analogs will be generically termed xe2x80x9cmutational vectorsxe2x80x9d. Such mutational vectors can introduce predetermined genetic changes into a target gene by a mechanism that is believed to involve homologous recombination and/or nucleotide excision and repair.
U.S. Pat. Nos. 5,565,350 and No. 5,731,181 to Kmiec describe mutational vectors that contain complementary strands wherein a first strand comprises ribonucleotide analogs that form Watson-Crick base pairs with deoxyribonucleotides of a second strand. U.S. Pat. No. 6,004,804 to Kumar and Metz describes certain improvements in duplex mutational vectors, including a variant in which the mutator region is present on only one of the two strands. The use of Kmiec type mutational vectors in mammalian systems is described in U.S. Pat. No. 5,760,012 and in conjunction with macromolecular carriers in International Patent Publication WO 98/49350 to Kren et al., and in related U.S. patent application Ser. No. 09/108,006. Additional descriptions of the use of Kmiec type mutational vectors can be found in Cole-Strauss et al., 1996, Science 273:1386; Kren et al., 1998, Nature Med. 4:285; and Bandyopadhyay et al., 1999, J. Biol. Chem. 274:10163.
The use of Kmiec type mutation vectors in plant cells is described in International Patent Publications WO 99/25853 to Pioneer Hi-Bred International, WO 99/07865 to Kimeragen, Inc. and WO 98/54330 to Zeneca Ltd. Scientific publications that describe the use of Kmiec type vectors in plants include Beetham et al., 1999, Proc. Natl. Acad. Sci. USA 96:8774 and Zhu, et al.,1999, Proc. Natl. Acad. Sci. USA 96:8768.
The use of Kmiec type mutational vectors and variants thereof, which are double stranded, is described in U.S. Pat. No. 6,004,804 to Kumar and Metz. The application of Kumar and Metz teaches, inter alia, that Kmiec type vectors and variants thereof can be used in bacterial cells.
The use of single stranded oligodeoxynucleotides as mutational vectors to effect changes in a chromosomal gene in the yeast, S. cerevisiae, was described in reports from the laboratory of Dr. F. Sherman, Yale University. Moerschell et al., 1988, Proc. Natl. Acad. Sci. USA, 85:524-528 and Yamamoto et al., 1992, Yeast 8:935-948. The optimum length of the mutational vectors used in these studies was 50 nucleotides.
An isolated report of the use of a 160 nucleotide single and double stranded polynucleotide to attempt to make alterations in a chromosomal gene can be found at Hunger-Bertling, 1990, Mol. Cell. Biochem. 92:107-116. The results for single stranded polynucleotides were ambiguous because only the product of the experiments using double-stranded polynucleotides were analyzed.
The use of single stranded DNA fragment of 488 base pairs to make specific genetic changes in the cystic fibrosis transmembrane conductance regulator gene has been reported by Goncz et al., 1998, Hum. Mol. Genetics 7:1913; and Kunzelmann et al., 1996, Gene Ther. 3:859.
Single stranded oligodeoxynucleotides of about 40 nucleotides in length in mammalian cells were used as a control for studies of episomal genes in which the oligodeoxynucleotide was covalently linked to a triplex forming oligonucleotide and that the oligodeoxynucleotide alone resulted in rates of predetermined genetic change of the episomal gene of about 1 per 5xc3x97104, or fewer. Chan et al., 1999, J. Biol. Chem. 74:11541. An earlier report of the use of single-stranded oligodeoxynucleotide to make predetermined changes in an episomal gene in a mammalian cell is found in Campbell et al., 1989, The New Biologist 1:223.
One aspect of the invention concerns oligodeoxynucleotides that have been modified by the attachment of an indocarbocyanine dye. Indocarbocyanine dyes are known as excellent fluorophores. The synthesis of blocked indocarbocyanine xcex2 cyanoethyl N,N-diisopropyl phosphoroamidites that are suitable for use in solid phase nucleotide synthesis is described in U.S. Pat. Nos. 5,556,959 and No. 5,808,044.
A second aspect of the invention concerns a composition comprising a single stranded oligonucleotide encoding a predetermined genetic change and a macromolecular carrier that comprises a ligand for a receptor on the surface of the target cell. A composition comprising a poly-L-lysine, a ligand for the asialoglycoprotein receptor and an antisense oligodeoxynucleotide of between 21 and 24 nucleotides is described in International Patent Publication WO 93/04701.
A third aspect of the invention concerns a modification of a oligodeoxynucleotide by the attachment of a 3xe2x80x2xe2x80x943xe2x80x2 linked nucleotide. U.S. Pat. No. 5,750,669 teaches such a modified oligodeoxynucleotide.
Citation or identification of any reference in Section 2, or any section of this application shall not be construed as an admission that such reference is available as prior art to the present invention.
The present invention is based on the unexpected discovery that single-stranded oligodeoxynucleotides, particularly when appropriately modified or placed in a composition with a suitable macromolecular carrier, can be as or more effective in making predetermined genetic changes to target genes in cells as the prior art, i.e., Kmiec type mutational vectors. A single stranded oligodeoxynucleotide suitable for use according to the present invention is termed hereafter a Single-Stranded Oligodeoxynucleotide Mutational Vector or a SSOMV.
In one embodiment the invention provides for a composition for use in making changes to the chromosomal genes of animal, e.g. mammalian, cells consisting of the oligodeoxynucleotide encoding the genetic change and a macromolecular carrier. The carrier can be either a polycation, an aqueous-cored lipid vesicle or a lipid nanosphere. In a further embodiment that is suitable for in vivo use, the carrier further comprises a ligand that binds to a cell-surface receptor that is internalized such as a lignad for a clathrin-coated pit receptor, e.g., the asialoglycoprotein receptor, the folic acid receptor or the transferin receptor. In preferred embodiments the oligodeoxynucleotide is modified by the attachment of 3xe2x80x2 and 5xe2x80x2 blocking substituents such as a 3xe2x80x2xe2x80x943xe2x80x2 linked cytosine nucleotide and a 5xe2x80x2 linked indocarbocyanine dye. In an alternative embodiment the modification can consist of the replacement of the 3xe2x80x2 most and/or 5xe2x80x2 most internucleotide phosphodiester linkage with a non-hydrolyzeable linkage such as a phosphorothioatediester linkage or a phosphoramidate linkage.
In a second embodiment the invention provides for the modification of the 3xe2x80x2 and 5xe2x80x2 end nucleotides of the oligodeoxynucleotide that encodes the predetermined genetic change. The invention is further based on the unexpected discovery that certain such modifications do not block the effectiveness of the oligodeoxynucleotide to produce genetic changes. One such embodiment is the combination of a 3xe2x80x2xe2x80x943xe2x80x2 linked cytosine nucleotide and a 5xe2x80x2 linked indocarbocyanine dye. So modified, the oligodeoxynucleotides are more than 50 fold more effective than a corresponding unmodified oligodeoxynucleotides when used to make genetic changes in bacterial cells.
In a third embodiment the invention provides compounds and methods for the introduction of a predetermined genetic change in a plant cell by introducing an oligodeoxynucleotide encoding the predetermined genetic change into the nucleus of a plant cell.
In preferred embodiments the oligodeoxynucleotide is modified by the attachment of 3xe2x80x2 and 5xe2x80x2 blocking substituents such as a 3xe2x80x2 -3xe2x80x2 linked cytosine nucleotide and a 5xe2x80x2 linked indocarbocyanine dye. In an alternative embodiment the modification can consist of the replacement of the 3xe2x80x2 most and 5xe2x80x2 most internucleotide phosphodiester linkage with a non-hydrolyzeable linkage such as a phosphorothioatediester linkage or a phosphoramidiate linkage. Alternatively, a 5xe2x80x2 linked indocarbocyanine dye and 3xe2x80x2 most internucleotide phosphodiester linkage a non-hydrolyzeable linkage can be used in yet a third embodiment.
The present invention may be understood more fully by reference to the following detailed description and illustrative examples of specific embodiments.
The sequence of the SSOMV is based on the same principles as prior art mutational vectors. The sequence of the SSOMV contains two regions that are homologous with the target sequence separated by a region that contains the desired genetic alteration, termed the xe2x80x9cmutator regionxe2x80x9d. The mutator region can have a sequence that is the same length as the sequence that separates the homologous regions in the target sequence, but having a different sequence. Such a mutator region causes a substitution. Alternatively, the homologous regions in the SSOMV can be contiguous to each other, while the regions in the target gene having the same sequence are separated by one, two or more nucleotides. Such a SSOMV causes a deletion from the target gene of the nucleotides that are absent from the SSOMV. Also, the sequence of the target gene that is identical to the homologous regions may be adjacent in the target gene but separated by one two or more nucleotides in the sequence of the SSOMV. Such an SSOMV causes an insertion in the sequence of target gene.
The nucleotides of the SSOMV are deoxyribonucleotides that are linked by unmodified phosphodiester bonds except that the 3xe2x80x2 terminal and/or 5xe2x80x2 terminal intemucleotide linkage or alternatively the two 3xe2x80x2 terminal and/or 5xe2x80x2 terminal internucleotide linkages can be a phosphorothioate or phosphorainidate. As used herein an intemucleotide linkage is the linkage between nucleotides of the SSOMV and does not include the linkage between the 3xe2x80x2 end nucleotide or 5xe2x80x2 end nucleotide and a blocking substituent, see below.
The length of the SSOMV depends upon the type of cell in which the target gene is located. When the target gene is a chromosomal gene of an animal cell, e.g., a mammalian or avian cell, the SSOMV is between 25 and 65. nucleotides, preferably between 31 and 59 deoxynucleotides and most preferably between 34 and 48 deoxynucleotides. The total length of the homologous regions is usually the length of the SSOMV less one, two or three nucleotides. A mutator nucleotide can be introduced at more than one position in the SSOMV, which results in more than two homologous regions in the SSOMV. Whether there are two or more homologous regions, the lengths of at least two of the homologous regions should each be at least 8 deoxynucleotides.
For prokaryotic cells, the length of the is SSOMV is between 15 and 41 deoxynucleotides. The preferred length of the oligodeoxynucleotide for prokaryotic use depends upon the type of 3xe2x80x2 protecting group that is used. When the 3xe2x80x2 protecting substituent is a 3xe2x80x2xe2x80x943xe2x80x2 linked deoxycytidine, the oligonucleotide is preferably between about 21 and 28 deoxynucleotides, otherwise the optimal length is between 25 and 35 deoxynucleotides. The lengths of the homology regions are, accordingly, a total length of at least 14 deoxynucleotides and at least two homology regions should each have lengths of at least 7 deoxynucleotides.
For plant cells, the length of the SSOMV is between 21 and 55 deoxynucleotides and the lengths of the homology regions are, accordingly, a total length of at least 20 deoxynucleotides and at least two homology regions should each have lengths of at least 8 deoxynucleotides.
Within these ranges the optimal length of the oligodeoxynucletide is determined by the GC content, the higher the GC content the shorter the optimal oligodeoxynucleotide. However, a GC content greater than 50% is preferred.
The SSOMV can be used with any type of animal cell, e.g., a mammalian cell, an avian cell, an insect cell, a fish cell, or a worm (nematode) cell. The SSOMV can also be used in any type of plant cell. Additionally, the SSOMV can be used with any type of bacterial cell, e.g., Gram-positive bacterial cells or Gram-negative bacterial cells. Exemplary types of bacteria include, Salmonella, E. coli, Pseudomonas, Rostani, etc. It is not important whether the cells are actively replicating or whether the target gene is transcriptionally active. However, when the target gene is located in a bacteria it is important that the bacteria be RecA+. Thus, most of the strains of bacteria commonly used in recombinant DNA work are not suitable for use in the present invention because such bacteria are RecAxe2x88x92 in order to reduce the genetic instability of the plasmids cloned therewith. Moreover, in bacterial cells the target gene can be located on a plasmid or on a bacterial artificial chromosome (BAC), as well as on the bacterial chromosome.
The SSOMV can be designed to be complementary to either the coding or the non-coding strand of the target gene. When the desired mutation is a substitution of a single base, it is preferred that the mutator nucleotide be a pyrimidine. To the extent that is consistent with achieving the desired functional result it is preferred that both the mutator nucleotide and the targeted nucleotide in the complementary strand be pyrimidines. Particularly preferred are SSOMV that encode transversion mutations, i.e., a C or T mutator nucleotide is mismatched, respectively, with a C or T nucleotide in the complementary strand.
In addition to the oligodeoxynucleotide the SSOMV can contain a 5xe2x80x2 blocking substituent that is attached to the 5xe2x80x2 terminal carbons through a linker. The chemistry of the linker is not critical other than its length, which should preferably be at least 6 atoms long and that the linker should be flexible.
The chemistry of the 5xe2x80x2 blocking substituent for mammalian, avian or plant cells is not critical other than molecular weight which should be less than about 1000 daltons. A variety of non-toxic substituents such as biotin, cholesterol or other steroids or a non-intercalating cationic fluorescent dye can be used. For use in bacterial systems, however, the blocking substituent has a major effect on the efficiency of the SSOMV and it is preferably a 3,3,3xe2x80x2, 3xe2x80x2-tetramethyl N,Nxe2x80x2-oxyalkyl substituted indocarbocyanine. Particularly preferred as reagents to make SSOMV are the reagents sold as Cy3(trademark) and Cy5(trademark) by Amersham Pharmacia Biotech, Piscataway, N.J., which are blocked phosphoroamidites that upon incorporation into an oligonucleotide yield 3,3,3xe2x80x2, 3xe2x80x2-tetramethyl N,Nxe2x80x2-isopropyl substituted indomonocarbocyanine and indodicarbocyanine dyes, respectively. When the indocarbocyanine is N-oxyalkyl substituted it can be conveniently linked to the 5xe2x80x2 terminal of the oligodeoxynucleotide through a phosphodiester with a 5xe2x80x2 terminal phosphate. The chemistry of the dye linker between the dye and the oligodeoxynucleotide is not critical and is chosen for synthetic convenience. When the commercially available Cy3 phosphoramidite is used as directed the resulting 5xe2x80x2 modification consists of a blocking substituent and linker together which are a N-hydroxypropyl, Nxe2x80x2-phosphatidylpropyl 3,3,3xe2x80x2, 3xe2x80x2-tetramethyl indomonocarbocyanine.
In an alternative embodiment, the indocarbocyanine dye, e.g., Cy3 phosphoramidate, can be linked to the oligodeoxynucleotide after the oligodeoxynucleotide has been synthesized.
In the preferred embodiment the indocarbocyanine dye is tetra substituted at the 3 and 3xe2x80x2 positions of the indole rings. Without limitation as to theory these substitutions prevent the dye from being an intercalating dye. The identity of the substituents at these positions are not critical.
The SSOMV can in addition have a 3xe2x80x2 blocking substituent. Again the chemistry of the 3xe2x80x2 blocking substituent is not critical, other than non-toxicity and molecular weight of less than about 1000, when the target gene is located in other than a bacterial cell. However, when the target gene is located in a bacterial cell the preferred 3xe2x80x2 blocking substituent is a so-called inverted nucleotide, i.e., a nucleotide that is linked by an unsubstituted 3xe2x80x2xe2x80x943xe2x80x2 phosphodiester, as is taught by U.S. Pat. No. 5,750,669. In a more preferred embodiment the inverted nucleotide is a thymidine or most preferred a deoxycytidine. For use in bacterial cells, the combination of a Cy3 5xe2x80x2 blocking substituent and an inverted deoxycytidine 3xe2x80x2 blocking substituent is particularly preferred as the two modifications have a synergistic effect on the efficacy of the SSOMV. The SSOMV with the above recited modifications can be synthesized by conventional solid phase nucleotide synthesis.
The SSOMV can be introduced into the cell containing the target gene by the same techniques that are used to introduce the Kmiec type mutational vectors into animal and plant cells. For bacterial cells, a preferred method of introducing the SSOMV is by electroporation.
For use with animal cells, including mammalian and avian cells, the preferred method of delivery into the cell is by use of a protective macromolecular carrier. Commercially available liposomal transfecting reagents such Lipofectamine(trademark) and Superfect(trademark) are designed so that the nucleic acid to be transfected is electrostatically adherent to the exposed surface of the liposome. Such carriers are not as preferred as protective macromolecular carriers. Suitable protective macromolecular carriers are disclosed in International Patent Publication WO 98/49350 and WO 99/40789 and in Bandyopadhyay et al., 1999, J. Biol. Chem. 274:10163, which are each hereby incorporated by reference in their entirety.
A particularly preferred macromolecular carrier is an aqueous-cored lipid vesicle or liposome wherein the SSOMV is trapped in the aqueous core. Such vesicles are made by taking a solvent free lipid film and adding an aqueous solution of the SSOMV, followed by vortexing, extrusion or passage through a microfiltration membrane. In one preferred embodiment the lipid constituents are a mixture of dioleoyl phosphatidylcholine/dioleoyl phosphatidylserine/ galactocerebroside at a ratio of 1:1:0.16. Other carriers include polycations, such as polyethylenimine, having a molecular weight of between 500 daltons and 1.3 Md, with 25 kd being a suitable species and lipid nanospheres, wherein the SSOMV is provided in the form of a lipophilic salt.
When the SSOMV are used to introduce genetic changes in mammalian and avian cells, it is preferred that the macromolecular carrier further comprise a ligand for a cell surface receptor that is internalized. Suitable receptors are the receptors that are internalized by the clathrin-coated pit pathway, such as the asialoglycoprotein receptor, the epidermal growth factor receptor and the transferin receptor. Also suitable are receptors that are internalized through the caveolar pathway such as the folic acid receptor. The galactocerebroside is a ligand for the asialoglycoprotein receptor. As used herein an internalizeable receptor is a receptor that is internalized by the clathrin-coated pit pathway or by the caveolar pathway.
The SSOMV can be used for any purpose for which the prior art mutational vectors were employed. Specific uses include the cure of genetic diseases by reversing the disease causing genetic lesion; such diseases includes for example hemophilia, xcex11 anti-trypsin deficiency and Crigler-Najjar disease and the other diseases that are taught by International Patent Publication WO 98/49350.
Alternatively, the SSOMV can be used to modify plants for the purposes described in patent publication WO 99/07865, which is hereby incorporated by reference in its entirety. An additional use of SSOMV in plants is the generation of herbicide resistant plants by means that avoid having to introduce a foreign or heterologous gene into a crop plant. Of particular interest is resistance to the herbicide glyphosate (ROUNDUP(copyright)). The identity of mutations that confer glyphosate resistance can be found in International Patent Publications WO 99/25853 and WO 97/04103.
Alternatively, the SSOMV can be used to modify bacteria. The use of SSOMV for the genetic manipulation of bacteria is particularly valuable in the fields of antibiotic production and in the construction of specifically attenuated bacteria for the production of vaccines. In both of the above applications it is important that antibiotic resistance genes not remain in the final modified bacteria.
Yet further, the SSOMV can be used in combination with a bacterial artificial chromosome (BAC) to modify a targeted gene from any species that has been cloned into a BAC. A fragment much larger than the targeted gene can be incorporated. The BAC having the cloned targeted gene is placed into a bacterial host and a predetermined genetic change is introduced according to the invention. A BAC subclone having the predetermined genetic change can be identified and the insert removed for further use. The present invention allows for the predetermined changes to be made without the time and expense attendant with obtaining making PCR fragments and inserting the fragments back into the original gene.